Mechanisms of Penile Erection and Basis for Pharmacological Treatment of Erectile Dysfunction (2011)

1. Nitric-Oxide Synthases in the Penis.

An important role for NO in the relaxation of CC smooth muscle and vasculature is widely accepted (Andersson and Wagner, 1995; Andersson, 2001; Musicki and Burnett, 2006; Musicki et al., 2009). There seems to be no doubt about the presence of nNOS in the cavernosal nerves and their terminal endings within the CC, and in the branches of the dorsal penile nerves and nerve plexuses in the adventitia of the deep cavernosal arteries (Andersson, 2001). Both the nerves (nNOS) and the endothelium (eNOS) of the CC may be the source of NO. The relative contribution of the different forms of NOS to erection has not been definitely established.

A variant of nNOS (penile nNOS) has been identified as two distinct isoforms in the penis of rat and mouse, a full-length α splice form and a β splice form that lacks the N-terminal postsynaptic density 95/disc-large/zona occludens domain, important for protein-protein interactions. Evidence suggests that the α splice variant is active in NO formation at nerve terminals, whereas the functional role of the β variant in vivo is unclear and might not be substantial (Magee et al., 1996; Gonzalez-Cadavid et al., 1999, 2000). The findings of Hurt et al. (2006) confirmed that alternatively spliced forms of nNOS are major mediators of penile erection. Mice lacking both eNOS and nNOS have erections, show normal mating behavior, and respond with erection to electrical stimulation of the cavernosal nerves. We were surprised to find that isolated corporal tissue from both wild-type and NOS-deleted animals showed similar responses to electrical stimulation (Burnett et al., 1996; Hurt et al., 2006). Functional studies support the occurrence and importance of eNOS in human cavernosal tissue (Andersson and Wagner, 1995; Musicki and Burnett, 2006), and this seems to be the case also in rat (Cartledge et al., 2000b) and mouse (Mizusawa et al., 2001) CC.

Although the interplay between the NOS isoenzymes continues to be a matter of study, existing evidence points toward a model (Fig. 4) in which nNOS initiates the erectile response, which is then maintained and increased by eNOS activity (the latter being activated by shear stress) (Hurt et al., 2002, 2006; Musicki and Burnett, 2006; Bivalacqua et al., 2007b; Musicki et al., 2009). eNOS has an indispensable role in the erectile response, and its activity and endothelial NO bioavailability are regulated by multiple post-translational molecular mechanisms, such as eNOS phosphorylation, eNOS interaction with regulatory proteins and contractile pathways, and actions of reactive oxygen species (ROS). These mechanisms regulate eNOS-mediated responses under physiological circumstances and provide various mechanisms whereby endothelial NO availability may be altered in states of vasculogenic erectile dysfunction (ED).

   

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Fig. 4.   

Cooperation between nNOS and eNOS. Existing evidence points toward a model in which nNOS initiates the erectile response, which is then maintained and increased by eNOS activity (the latter being activated by shear stress). [Modified from Hurt KJ, Musicki B, Palese MA, Crone JK, Becker RE, Moriarity JL, Snyder SH, and Burnett AL (2002) Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection. Proc Natl Acad Sci USA 99:4061–4066. Copyright © 2002 National Academy of Sciences, USA. Used with permission.].

The influence of androgens on erectile function might to an important extent be mediated by the NO/cGMP pathway (Andersson, 2001) even if non–NO-dependent pathways have been demonstrated (Reilly et al., 1997; Mills and Lewis, 1999; Mills et al., 1999). Castration of rats and treatment with the antiandrogen flutamide reduced constitutive penile NOS activity (Chamness et al., 1995; Lugg et al., 1996; Penson et al., 1996).

Compared with young rats, NOS-containing nerves, NOS mRNA expression, and NOS activity decreased in old animals (Garban et al., 1995; Carrier et al., 1997; Dahiya et al., 1997). ED associated with, for example, diabetes was found to be associated by a decreased nNOS content and activity in the rat CC (Vernet et al., 1995; Autieri et al., 1996; Rehman et al., 1997). In humans, the diabetic ED was suggested to be related to the effects of advanced glycation end products on NO formation (Seftel et al., 1997). Cartledge et al. (2000a) found in rats that glycosylated human hemoglobin impaired CC smooth muscle relaxation by generation of superoxide anions and extracellular activation of NO.

Angulo et al. (2006), evaluating the influence of PKC activity on penile smooth muscle tone in tissues from diabetic and nondiabetic men with ED, found that overactivity of PKC in diabetes is responsible for enhanced contraction and reduced eNOS-dependent relaxation of human CC smooth muscle.

2. Guanylyl Cyclases.

The GCs, comprising both membrane-bound (particulate, pGC) and soluble isoforms (sGC), are expressed in nearly all cell types (Lucas et al., 2000). The GCs are stimulated by NO, natriuretic peptides, and other endogenous ligands (e.g., CO). CO, generated via heme oxygenase-mediated degradation of cellular heme, also stimulates sGC, albeit to a lesser extent than NO (Friebe et al., 1996).

Kim et al. (1998) demonstrated production of cGMP by pGC in the CC membranes of rabbit and rat stimulated by C-type natriuretic peptide 1–22 (CNP), atrial natriuretic peptide 1–28 (ANP), and brain natriuretic peptide 1–26 (BNP). In addition, CNP, but not ANP, relaxed precontracted isolated preparations of rabbit CC. Aizawa et al. (2008) investigated the effects of ANP, BNP, and CNP on intracavernosal pressure and systemic blood pressure in conscious, free-moving rats. They found that erectile responses could be initiated by ANP, by BNP, and less effectively by CNP. ANP and BNP have a high affinity for GC-A, suggesting that this receptor is involved in the responses.

Küthe et al. (2003) studied the expression of GC-B, a receptor of CNP in the human CC. mRNA transcripts were detected encoding for GC-B, and the expression was verified at the protein level by immunohistochemistry that demonstrated GC-B in CC and helical artery smooth muscle cells. CNP increased intracellular cGMP. In organ bath studies with CC muscle strips, CNP caused smooth muscle relaxation. It was concluded that CNP and its receptor might have a role in the induction of penile erection. A relaxant effect of ANP and uroguanylin was demonstrated in strips of human CC by Sousa et al. (2010). They found that uroguanylin relaxed the strips by a GC and K_Ca-channel-dependent mechanism and suggested that the natriuretic peptide receptors are potential targets for the development of new drugs for the treatment of ED. However, in the penis, sGC is probably the most important receptor for NO as a signaling molecule. The enzyme, which catalyzes the conversion of GTP into cGMP, consists of two different subunits and contains a prosthetic heme group that mediates up to 400-fold activation by NO. Nimmegeers et al. (2008) assessed the functional importance of the sGCα₁β₁ isoform in CC from male sGCα₁(−/−) and wild-type mice. The relaxation to endogenous NO (from acetylcholine, bradykinin, and electrical field stimulation) was nearly abolished in the sGCα₁(−/−) CC. In the sGCα₁(−/−) mice, the relaxing influence of exogenous NO (from sodium nitroprusside and NO gas), 3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (BAY 41-2272; NO-independent sGC stimulator), and methyl-(2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl))-3-isoquinoline carboxylic acid, sulfate salt (T-1032; phosphodiesterase type 5 inhibitor) were also significantly decreased. It was concluded that the sGCα₁β₁ isoform is involved in CC smooth muscle relaxation in response to NO and NO-independent sGC stimulators.

3-(5′-Hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) was shown to elicit a direct activation of sGC (Fig. 5) by allosterically increasing the affinity for GTP and increasing the maximal enzyme activity, leading to increased cGMP levels in smooth muscle cells (Mülsch et al., 1997; Friebe and Koesling, 1998). Moreover, YC-1 caused a large activation in the presence of the NO donor sodium nitroprusside, which led to a remarkable 2200-fold stimulation of the human recombinant sGC (Lee et al., 2000). YC-1 caused concentration-dependent relaxant responses in NA-contracted rat CC preparations, and enhanced responses to electrical field stimulation. YC-1 also enhanced the relaxant response induced by carbachol. In vivo, YC-1 not only elicited dose-dependent erectile responses when administered intracavernosally but also increased the effects on intracavernosal pressure produced by stimulation of the cavernosal nerve (Mizusawa et al., 2002a). YC-1 was able to significantly augment the pro-erectile effects of a suboptimal dose of apomorphine (Hsieh et al., 2003).

   

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Fig. 5.   

Soluble guanylyl cyclase exists as a heterodimer of α (82 kDa) and β (70 kDa) subunits and has a catalytic site and two allosteric sites. One allosteric site is defined by the NO binding site (the heme; Fe), and the second is represented by the binding of YC-1. Agents such as YC-1 can activate sGC after binding to the allosteric site in the enzyme, leading to an increase in the intracellular concentration of cGMP, relaxation of cavernosal tissue, and facilitation of penile erection in vivo.

The pyrazolopyridine derivative BAY41-2272 was also found to stimulate sGC in a NO-independent manner and caused concentration-dependent relaxation of human and rabbit cavernosum (Kalsi et al., 2003) but induced only weak penile erections in conscious rabbits after intravenous and oral administration in the absence of an NO donor. However, the efficacy of BAY 41-2272 was potentiated by the simultaneous administration of SNP (Bischoff et al., 2003). In addition, other GC activators have been studied (Lasker et al., 2010). It has been pointed out that the efficacy of these sGC activators for ED has not been determined and that pilot studies are needed to assess their utility in the treatment of this disease. The use of both sGC activators and stimulators may be useful in conditions of altered heme conformation as well as in conditions in which NO synthesis is impaired (Lasker et al., 2010).

3. cGMP Signaling.

The mechanisms involved in cGMP signaling have recently been extensively reviewed by Francis et al. (2010). Stimulation of GCs by NO and natriuretic peptides, and other endogenous ligands (e.g., CO), generates cGMP, which has influence on a number of vascular cell types and regulates vasomotor tone, endothelial permeability, cell growth, and differentiation, as well as platelet and blood cell interactions. There is evidence for reciprocal regulation of the NO-cGMP and natriuretic peptide-cGMP pathways and that one cGMP generating system may compensate for the dysfunction of the other (Kemp-Harper and Schmidt, 2009; Francis et al., 2010). cGMP signals via three main receptors in eukaryotic cells: ion channels, phosphodiesterases, and protein kinases (Lucas et al., 2000; Kemp-Harper and Schmidt, 2009; Francis et al., 2010). The molecular targets, which are activated by cGMP and finally produce relaxation of penile smooth muscle and erection, are still only partly known.

Three different cGMP-dependent protein kinases (cGKIα, cGK1β, and cGKII; also named PKGIα, PKGIβ, and PKGII) have been identified in mammals. Inactivation of cGKI in mice abolished both NO/cGMP-dependent relaxation of vascular and intestinal smooth muscle and inhibition of platelet aggregation, causing hypertension, intestinal dysmotility, and abnormal hemostasis (Pfeifer et al., 1998). CGKI-deficient [cGKI(−/−)] mice show a very low ability to reproduce. In CC tissue from these mice, the relaxant response to neuronally or endothelially released or exogenously administered NO was markedly reduced (Hedlund et al., 2000a). The expression of cGKI in penile tissue from cGKI(+/+) mice, as revealed by immunohistochemistry, was confined to the smooth muscle of the walls of the central and helicine arteries and to the smooth muscle of the trabecular septa surrounding the cavernosal spaces. This is in line with its presumed role in the erectile events. The total innervation (protein gene product 9.5 immunoreactivity) and distribution of nerve populations containing transmitters or transmitter-forming enzymes believed to be important in the regulation of tone in CC tissue (Andersson and Wagner, 1995) were similar in normal and cGKI-null mice (Hedlund et al., 2000).

Analysis of the NO/cGMP-induced relaxation clearly showed that cGKI is the major mediator of the cGMP signaling cascade in CC tissue. Its absence cannot be compensated for by the cAMP signaling cascade, which relaxes normal and cGKI-null penile erectile tissue to a similar extent. Taken together, these findings suggest that activation of cGKI is a key step in the signal cascade leading to penile erection.

The expression of cGKI was examined in CC specimens from patients with and without ED (Klotz et al., 2000). In all specimens of cavernosal tissue, a distinct immunoreactivity was observed in different parts and structures, with a high expression in smooth muscle cells of vessels and in the fibromuscular stroma. No clear immunoreactivity against cGKI was found in the endothelium. There was no distinct difference in immunoreactivity and cellular distribution between potent and impotent patients. This does not exclude that dysfunction of cGKI can be a cause of ED in humans, and that cGKI can be an interesting target for pharmacological intervention.

Bivalacqua et al. (2007a) investigated the expression of cGKIα (PKGIα) and cGKIβ (PKGIβ) in the CC and evaluated the effect of adenoviral gene transfer of cGKIα to the erectile compartment on EF in a rat model of diabetes. They found cGKIα and cGKIβ activities to be reduced in the erectile tissue of the diabetic rat. Supporting the role of cGK in the erectile process, gene transfer of cGKIα to the penis restored cGK activity and erectile function in vivo.

4. Phosphodiesterases.

PDEs catalyze the hydrolysis of the second messengers cAMP and cGMP, which are involved in the signal pathways most important for relaxation of CC smooth muscle. The protein superfamily of cyclic nucleotide PDEs can be subdivided into at least 11 families of structurally and functionally related enzymes. More than 50 isoforms have been characterized so far, all differing in their primary structures, specificity for cAMP and cGMP, cofactor requirements, kinetic properties, mechanisms of regulation, and tissue distributions (Francis et al., 2010). Because of their central role in smooth muscle tone regulation and the considerable variation of PDE isoenzymes with respect to species and tissues, PDEs have become an attractive target for drug development. In human cavernosal tissue, at least 13 isoenzymes have been identified, including PDE3 (cGMP-inhibited cAMP PDE), PDE4 (cAMP-specific PDE), and PDE5 (cGMP-specific PDE) (Küthe et al., 1999, 2000, 2001). Functionally, PDE 3A and 5A seem to be the most important (Küthe et al., 1999, 2000, 2001; Francis et al., 2010). Three of the PDE families (PDEs 5, 6, 9) have a >100-fold substrate preference for cGMP over cAMP as substrate and are therefore considered to be “cGMP-specific PDEs.” PDE5 and PDE9 are the only “cGMP-specific PDEs” that are expressed in peripheral tissues; PDE6 is expressed in the retina (Francis et al., 2010).

PDE5, which is present in high concentrations in the smooth muscle of the CC, is the most therapeutically important because it is the target for the currently most widely used ED drugs, the PDE5 inhibitors. PDE5 is a homodimer containing two identical subunits with molecular mass of approximately 100,000 Da per subunit. Each of the two subunits has a catalytic domain and a regulatory domain. The catalytic domain, which is the target of PDE5 inhibitors, contains a single binding site for cGMP (Francis et al., 2010). Because they have structures similar to that of cGMP, sildenafil or other PDE5 inhibitors can also occupy the catalytic site, thus blocking access to cGMP competitively. However, sildenafil occupies the site approximately 1000 times more avidly than does cGMP, with the result that cGMP cannot bind to gain access to the catalytic machinery and accumulates in the smooth muscle cells of the CC. This leads to relaxation of the CC and penile erection (Francis et al., 2010). It is noteworthy that PDE5 seems to regulate sGC-derived but not pGC-derived cGMP, because ANP-mediated vasodilation is not enhanced in vitro or in vivo by the PDE5 inhibitor sildenafil (Kemp-Harper and Schmidt, 2009).

Lin et al. (2000, 2002, 2005) reported cloning of three PDE5 isoforms from human penile tissues. Two of the isoforms were identical to PDE5A1 and PDE5A2, respectively, which had previously been isolated from nonpenile tissues. The third isoform was novel and was called PDE5A3; this isoform was confined to tissues with a smooth muscle or cardiac muscle component.

The identification of the various PDE families has been paralleled by the synthesis of selective or partially selective inhibitors. Sildenafil, vardenafil, and tadalafil are highly selective inhibitors of PDE type 5, but several new compounds have been developed, some of which have significantly different structures (Boolell et al., 1996a,b; Francis and Corbin, 2005; Francis et al., 2010). They all enhance NO-mediated relaxation of CC from various species in vitro and in vivo by increasing the intracellular concentrations of cGMP by amplifying the endogenous NO-cGMP pathway (Kouvelas et al., 2009; Francis et al., 2010). The molecular mechanisms involved have been reviewed in detail elsewhere (Francis et al., 2010). PDE5 inhibitors are currently first-line treatment of ED (see section VIII.C), and several new selective PDE5 inhibitors are in different stages of development or have already been introduced clinically (Hatzimouratidis and Hatzichristou, 2008; Dorsey et al., 2010; Eardley et al., 2010).

Androgenic influences may affect PDE5 function in the penis (Morelli et al., 2004; Traish and Kim, 2005; Zhang et al., 2005), but the effect may be indirectly mediated by the influence of androgens on NOS function. These insights indicate that regulatory factors of PDE5 expression and activity in the penis critically determine the biological role of the enzyme.

5. Other Gaseous Mediators.

Carbon monoxide (CO) and hydrogen sulfide (H₂S) are, together with NO, considered the principal peripheral proerectile gaseous transmitters that can be released mainly by cholinergic nerves and the sinusoidal endothelium to relax CC smooth muscle through the cGMP pathway. As mentioned, CO is generated via HO-mediated degradation of cellular heme and is able to stimulate sGC, but to a lesser extent than NO (Friebe et al., 1996).

A significant, positive effect of the HO/CO system on penile erection has been reported in several studies, and its potential role as molecular target in treatment of ED was reviewed by Shamloul (2009). None of the studies examined the role of the HO/CO system in aging animals, aging being considered the most important risk factor for ED. It was concluded that the HO/CO system might have a role in penile erection. However, further studies are needed to precisely delineate the importance of the HO/CO system in the physiology and pathophysiology of male function and sexual dysfunction.

l-Cysteine acts as a natural substrate for the synthesis of H₂S. Exogenous H₂S [administered as sodium hydrogen sulfide (NaHS)] or l-Cys caused a concentration-dependent relaxation of strips of human CC. In rats, the intracavernosal administration of either NaHS or l-Cys elicited penile erection (d’Emmanuele di Villa Bianca et al., 2010). These observations were suggested to indicate that a functional l-Cys/H₂S pathway may be involved in mediating penile erection in humans and other mammals.

1. Adrenomedullin, Calcitonin Gene-Related Peptide, Nociceptin.

Adrenomedullin, a peptide belonging to the CGRP family, was first isolated from human phaeochromocytoma and has been suggested to serve as a circulating hormone regulating systemic arterial pressure (CGRP; Kitamura et al., 1993). The peptide has been demonstrated in the endothelial cells of human cavernosal tissue (Marinoni et al., 2005). Adrenomedullin inhibits the contraction of several types of smooth muscle cells not only through an increase in cAMP production, but also be stimulating the release of NO (Miura et al., 1995). Injected intracavernosally in cats, adrenomedullin caused increases in intracavernosal pressure and in penile length, an effect also found with CGRP (Champion et al., 1997a,b,c). Because the erectile responses to adrenomedullin or CGRP were unaffected by NOS inhibition with l-NAME or by K_ATP channel inhibition with glibenclamide, it was suggested that NO or K_ATP channels were not involved in the responses. The CGRP antagonist CGRP (8–37), at doses having no effect on the adrenomedullin response, reduced the responses to CGRP, suggesting that the peptides acted on different receptors. In the highest doses used, both adrenomedullin and CGRP reduced blood pressure (Champion et al., 1997a,b,c). Similar effects were found when adrenomedullin was injected intracavernosally in rats (Nishimatsu et al., 2001). In isolated precontracted rabbit CC strips, adrenomedullin caused a concentration-related relaxant effect (Gokce et al., 2004).

Bivalacqua et al. (2001a) used adenoviral gene transfer of prepro-CGRP to restore erectile function in the aged rat and found an improvement in erectile function. CGRP had been suggested previously to have potential for treating ED (Stief et al., 1990, 1991; Truss et al., 1994), but it does not seem likely that either adrenomedullin or CGRP will be clinically useful for treatment of ED.

Nociceptin is a 17-amino acid peptide that shares structural homology with the dynorphin family of peptides. It differs from other opioid peptides in that it does not have the NH₂-terminal residue, which is essential for activity at μ, δ, and κ opioid receptors (Henderson and McKnight, 1997; Calo’ et al., 2000). The peptide is an endogenous ligand for the orphan opioid receptor that has been identified in several species: the human clone is called ORL1. It seems to be involved in a diversity of functions (Chiou et al., 2007) and has been shown to interfere with (inhibit) dopamine release in the brain (Olianas et al., 2008). Whether the latter action has any effects on erectile mechanisms or sexual behavior is not known.

Champion et al. (1997a) compared the erectile responses to intracavernosal nociceptin administration with those of a triple drug combination, VIP, adrenomedullin, and an NO-donor in cats. Nociceptin in doses of 0.3 to 3 nM elicited dose-related increases in intracavernosal pressure and penile length comparable with that of the triple drug combination, but the duration of the response was shorter. It does not seem likely that nociceptin has an important role in erectile mechanisms or that the ORL1 receptor is a reasonable target for drugs improving erectile function.

2. Endocannabinoids.

There is little information concerning the peripheral effect of cannabinoids on CC tissue. Ghasemi et al. (2006) investigated the effect of the endogenous cannabinoid anandamide on the NANC relaxant responses to electrical field stimulation in isolated rat CC. They showed that anandamide has a potentiating effect on NANC-mediated relaxation through both CB1 and vanilloid receptors. Furthermore, they demonstrated that the NO-mediated component of the NANC relaxant responses to electrical stimulation is involved in this enhancement. The same group studied the effect of biliary cirrhosis on NANC-mediated relaxation of rat CC and the possible roles of endocannabinoid and NO systems in this model (Ghasemi et al., 2007). NANC-mediated relaxation was enhanced in CC strips from cirrhotic animals. Anandamide potentiated the relaxations in both groups. Either 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM251; CB1 receptor antagonist) or capsazepine (transient receptor potential vanilloid 1 receptor antagonist), but not 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl-(4-methoxyphenyl)methanone (AM630; CB2 receptor antagonist), prevented the enhanced relaxations of cirrhotic strips. Both the nonselective NOS inhibitor l-NAME and the selective neuronal NOS inhibitor N^ω-propyl-l-arginine inhibited relaxations in both groups, but the cirrhotic groups were more resistant to the inhibitory effects of these agents. Relaxations in response to sodium nitroprusside (NO donor) were similar in tissues from the two groups. The authors concluded that cirrhosis potentiates the neurogenic relaxation of rat CC, probably via the NO pathway and involving cannabinoid CB1 and transient receptor potential vanilloid 1 receptors.

Western blotting of CC tissues have demonstrated the existence of CB1 receptors in CC strips of rat and rhesus monkey (Gratzke et al., 2010b). In contrast to rat corporal tissue, relaxant responses to electrical stimulation were not altered in the presence of anandamide at 10 nM to 30 μM in monkey CC strips. Further studies are needed to elucidate the role of the endocannabinoid system in erectile tissue.

3. Tumor Necrosis Factor-α.

Reactive oxygen species are important mediators of endothelial cell injury and dysfunction, which are the major triggers of pathophysiological processes leading to CV disease. A candidate factor in causing ROS production in endothelial cells is tumor necrosis factor α (TNF-α). TNF-α has been shown to play an important role in CV disease, mainly because of its direct effects on the vasculature (Chen et al., 2008; Zhang et al., 2009a), and may also be involved in ED, because high levels of TNF-α were demonstrated in patients with ED (Carneiro et al., 2010). TNF-α KO mice were found to exhibit changes in cavernosal reactivity that would facilitate erectile responses. Thus the animals showed decreased responses to adrenergic nerve stimulation and increased NANC- and endothelium-dependent relaxation that were associated with increased corporal eNOS and nNOS protein levels (Carneiro et al., 2009). TNF-α impaired endothelium-dependent and NO-mediated vasodilation in various vascular beds (Chen et al., 2008; Zhang et al., 2009a), and a key role for TNF-α in mediating endothelial dysfunction in ED has been suggested (Carneiro et al., 2009). Blockade of TNF-α actions (which is clinically possible) may theoretically represent an alternative therapeutic approach for erectile dysfunction, especially in pathological conditions associated with increased levels of this cytokine. Whether TNF-α can be a treatment alternative in such cases of ED remains to be established.

However, treatment with TNF-α antagonists may not be without problems. Experiences from treatment of disorders other than ED have shown that a third of patients do not respond to treatment for various reasons (Desroches et al., 2010).

In Vivo.

CC tissue evokes electric waves that seem to be of diagnostic significance in evaluation of ED. However, it has been difficult to diagnose cavernosal nerve impairment in patients. Electrocavernosography has the potential to function as an investigative tool in diagnosing different types of ED (Wagner et al., 1989; Sasso et al., 1996; Shafik et al., 2004a,b). Advances in clinical electrophysiology now allow for consistent recording of intrapenile electrical activity. One promising method is evoked cavernosal activity, which can be recorded after a brief, startling stimulus (Yang et al., 2008; Yilmaz et al., 2009).

In Vitro.

A variety of ion channels have been identified in CC smooth muscle cells, but there have been few electrophysiological investigations of whole corporal smooth muscle preparations (Andersson, 2001). In the proximal part of the rat corpus spongiosum (penile bulb), Hashitani (2000) demonstrated spontaneous action potentials in the inner muscle layer. On the other hand, no action potentials could be detected by electrophysiological investigation of cultured human CC smooth muscle cells (Christ et al., 1993). If this is valid also for the cells in vivo, it calls for an alternative mechanism for impulse propagation. Gap junctions may provide such a mechanism.

As underlined by Christ (2000), signal transduction in corporal smooth muscle is more a network event than the simple activation of a physiological cascade or pathway in individual myocytes. Gap junctions may contribute to the modulation of corporal smooth muscle tone and, thus, erectile capacity, and intercellular communication through gap junctions can provide the corpora with a significant “safety factor” or capacity for plasticity/adaptability of erectile responses.

Coordination of activity among the corporal smooth muscle cells is an important prerequisite to normal erectile function. The autonomic nervous system plays an important role in this process by supplying a heterogeneous neural input to the penis. For example, the activity of the various parts of the autonomic nervous system differs dramatically during erection, detumescence, and flaccidity (Becker et al., 2000). The role of the autonomic nervous system in normal penile function must be coordinated; the firing rate of the autonomic nervous system, myocyte excitability, signal transduction processes, and the extent of cell-to-cell communication between corporal smooth muscle cells must be carefully integrated to ensure normal erectile function.

1. The BK_Ca Channel.

The BK_Ca channel has been well characterized in both human and rat corporal smooth muscle (Wang et al., 2000; Archer, 2002). BK_Ca channels are formed by two subunits: a pore-forming α-subunit and a modulatory transmembrane β-subunit. BK_Ca channel mRNA and protein have been detected in both freshly isolated human corporal tissues and cultured corporal smooth muscle cells (Christ et al., 1999). Consistent with such observations, the single-channel conductance (≈180 pS), whole-cell outward currents, and voltage and calcium sensitivity of the K_Ca channel are remarkably similar when comparing data collected with patch clamp techniques on freshly isolated corporal smooth muscle myocytes versus similar experiments on short-term explant cultured corporal smooth muscle cells (Fan et al., 1995; Lee et al., 1999a,b).

The BK_Ca channel seems to be an important convergence point in modulating the degree of corporal smooth muscle contraction. The activity of this channel is increased after cellular activation of either the cAMP pathway by 8-bromo-cAMP or PGE₁ (Lee et al., 1999a) or the cGMP pathway by 8-bromo-cGMP (Wang et al., 2000). It seems clear that the two most physiologically relevant endogenous second-messenger pathways act to modulate corporal smooth muscle tone (i.e., elicit relaxation), at least in part, via activation of the BK_Ca channel subtype. The resulting hyperpolarization, in turn, is coupled to decreased transmembrane calcium flux through L-type VDCCs and, ultimately, smooth muscle relaxation.

The functional role of BK channels in the CC was investigated by Werner et al. (2005), using a knock-out mouse lacking the Slo gene (Slo(−/−)) responsible for the pore-forming subunit of the BK channel. CCSM strips from Slo(−/−) mice demonstrated a four-fold increase in phasic contractions, in the presence of phenylephrine. Nerve-evoked relaxations of precontracted strips were reduced by 50%, both in strips from Slo(−/−) mice and by blocking BK channels with iberiotoxin in the Slo+/+ strips. Consistent with the in vitro results, in vivo intracavernosal pressure exhibited pronounced oscillations in Slo(−/−) mice, but not in Slo+/+ mice. Furthermore, intracavernosal pressure increases to nerve stimulation, in vivo, were reduced by 22% in Slo(−/−)mice. These results indicate that the BK channel has an important role in erectile function, and loss of the BK channel leads to erectile dysfunction. Supporting this view, intracavernosal injection of cDNA encoding the human BK channel led to a reversal of erectile dysfunction in aged or diabetic rats and in atherosclerotic monkeys (Christ et al., 1998; Christ et al., 2004, 2009). These studies support the idea that elevating BK_Ca-channel expression can restore erectile function after age- or disease-induced decline. Opening of BK_Ca channels would be an alternative way of restoring an insufficient erectile function (e.g., Boy et al., 2004). Kun et al. (2009) found that NS11021 (1-(3,5-Bis trifluoromethyl- phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea), a novel opener of BK_Ca channels caused relaxation of both intracavernosal arteries and CC strips primarily through opening of BK_Ca channels. It was also effective in facilitating erectile responses in anesthetized rats. These results suggest a potential for use of BK_Ca openers in the treatment of ED. However, so far no successful clinical results have been published.

2. The K_ATP Channel.

Western blots on isolated tissue strips, and immunocytochemistry of cultured corporal smooth muscle cells, using antibodies to the K_ATP channel, have documented the presence of the K_ATP channel protein (Christ et al., 1999). K_ATP channels are thought to be composed of two proteins: (1) an inward-rectifying K+ channel subunit (Kir; serving as the channel pore) and (2) a sulfonylurea receptor (SUR). In the human corporal smooth muscle the K_ATP channel are composed of the heteromultimers of Kir6.1 and Kir6.2 (Insuk et al., 2003). Experiments on freshly isolated corporal smooth muscle cells have documented the presence of two distinct ATP-sensitive K⁺ currents in cultured and freshly dissociated human corporal smooth muscle cells (Lee et al., 1999a). Consistent with observations at the single channel level, whole cell patch clamp studies documented a significant, glibenclamide-sensitive, increase in the whole cell outward K⁺ currents in the presence of the K channel modulator levcromakalim (see Lee et al., 1999a). These data, ranging from the molecular, through the cellular and whole tissue levels, clearly document the presence and physiological relevance of the K_ATP channel subtype(s) to the modulation of human corporal smooth muscle tone. Several studies have documented that K channel modulators, putative activators of the K_ATP channel, elicit a concentration-dependent relaxation of isolated human corporal smooth muscle (Andersson, 1992, 2001). Although activation of the K_ATP channel has been suggested to be involved in the action of yohimbine (Freitas et al., 2009), phentolamine (Silva et al., 2005) and testosterone (Yildiz et al., 2009) on cavernosal tissue, the importance of this contribution to the total effects of these agents on erectile mechanisms has not been established.

3. Calcium Channels.

The distribution of Ca²⁺ ions across the CC smooth muscle cell membrane ensures that opening of Ca²⁺ channels will lead to influx of Ca²⁺ ions into the CC smooth muscle cell down their electrochemical gradient. The movement of positive charge into the smooth muscle cell has the opposite effect of the movement of K⁺ out of the cell, and therefore, will lead to depolarization. Several studies have documented the importance of continuous transmembrane Ca²⁺ influx through L-type voltageVDCCs to the sustained contraction of human CC smooth muscle (Andersson, 2001). There seems to be only one published report of inward Ca²⁺ currents in CC smooth muscle using direct patch clamp methods (Noack and Noack, 1997). However, much of the most compelling mechanistic data concerning the role of Ca²⁺ channels in modulating human CC smooth muscle tone have been established using digital imaging microscopy of Fura-2 loaded cultured CC smooth muscle cells. These studies have provided strong evidence for the presence and physiological relevance of transmembrane Ca²⁺ flux through the L-type voltage-dependent calcium channel in response to cellular activation with e.g., ET-1 (ET_A/B receptor subtype) and phenylephrine (α₁-adrenergic receptor subtype)(Christ et al., 1992b; Zhao and Christ, 1995; Staerman et al., 1997). Nifedipine-sensitive Ca²⁺ currents have been recorded from isolated rabbit CC smooth muscle cells (Craven et al., 2004), suggesting that the individual cells may be capable of generating action potentials by the opening of L-type Ca²⁺ channels.

The fact that inhibitors of L-type voltage-dependent calcium channels inhibit spontaneous contractions in isolated strips of CC is consistent with the idea that Ca²⁺ influx via this pathway is important for tone generation. However, the ability of human, rabbit and rat CC to develop phasic contractions and phasic electrical activity (e.g., Hashitani et al., 2005), suggests that the intracellular Ca²⁺ levels are oscillatory. This was confirmed in a study by Sergeant et al. (2009) in which spontaneous Ca²⁺ waves were shown to be generated both in individual smooth CC muscle cells and across intact slices of CC tissue, where bursts of Ca²⁺ signals could be seen to trigger both phasic and tonic components of contraction. This “pacemaker” activity is likely to be of primary importance to the normal function of the CC, as it was shown to be associated with tissue contraction and inhibited by the NO/cGMP pathway.

McCloskey et al. (2009) studied VDCCs in rabbit CC myocytes dispersed enzymatically for patch clamp recording and confocal Ca²⁺ imaging. They found that the isolated myocytes developed robust VDCCs that could be separated into two components, one an L-type Ca²⁺current, and the other a putative T-type current. The L-current facilitated conversion of local Ca²⁺ events into global Ca²⁺ waves, whereas the putative T-current seemed to play little part in this process. These findings confirm those of Zeng et al. (2005) demonstrating that human CC cells express T-type (α2G) Ca²⁺ channels that are involved in maintaining [Ca²⁺] homeostasis.

4. Chloride Channels.

The contribution of chloride channels/currents to the modulation of human corporal smooth muscle tone is less well understood than that of the other ion channels. Studies of calcium-activated Cl⁻ (Cl_Ca) function in the smooth muscle of CC have shown these channels to be involved in both the maintenance of spontaneous tone and the contractile response to noradrenaline and other agonists (Fan et al., 1999; Karkanis et al., 2003; Craven et al., 2004; Williams and Sims., 2007; Chu and Adaikan, 2008; Chung et al., 2009b). Karkanis et al. (2003) demonstrated an excitatory calcium Cl_Ca current in both human and rat corporal myocytes. This current was activated by agonist-induced Ca²⁺ release from stores and also occurs as spontaneous transient currents, which are typically caused by Ca²⁺ sparks. Cl_Ca channel blockers enhanced and prolonged the rise in pressure after cavernosal nerve stimulation, indicating that Cl⁻ current contributes to the regulation of intracavernosal pressure. Craven et al. (2004) proposed that Ca²⁺ activated Cl⁻ currents underlie detumescent tone in the CC, and modulation of this mechanism by the NO-cGMP pathway is important during penile erection. In support of these observations, Williams and Sims (2007) demonstrated that that Ca²⁺ sparks in CC arise from Ca²⁺ release through ryanodine receptors and give rise to Cl_Ca current. They also showed physiological regulation of spark frequency with depolarization as a result of voltage-dependent Ca²⁺ entry. These results were confirmed by Sergeant et al. et al. (2009), revealing that each of the Ca²⁺ waves was associated with an inward current typical of the Ca^2+-activated Cl⁻ currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca²⁺ store, because they were blocked by cyclopiazonic acid and agents that interfere with ryanodine receptors and IP₃-mediated Ca²⁺ release. Chu and Adaikan (2008) underlined the importance of Cl⁻ currents as a mechanism in the maintenance of CC tone produced by adrenergic and various endogenous constrictors in strips of rabbit CC. They suggested that the modulation of Cl⁻¹ current could be an attractive and effective approach to regulate penile erection. In rats, Chung et al. (2009b) performed an in vivo study of the functional role of chloride channels in regulating erectile activity and concluded that chloride channels may play an important role in the regulation of CC tone.

1. Contraction.

In general, smooth muscle contraction is controlled by Ca²⁺ and Rho kinase signaling pathways (Berridge, 2008). Changes in the sarcoplasmic Ca²⁺ concentration, and thereby in the contractile state of the smooth muscle cell, can occur with or without changes in the membrane potential (Somlyo and Somlyo, 1994, 2000; Stief et al., 1997; Berridge, 2008) (Fig. 6). Action potentials or long-lasting changes in the resting membrane depolarize the membrane potential, thus opening voltage-gated L-type Ca²⁺ channels (Kuriyama et al., 1998). Thus, Ca²⁺ enters the sarcoplasm driven by the concentration gradient and triggers contraction. Membrane channels other than Ca²⁺ channels may also induce changes in the membrane potential. Opening of K⁺ channels can produce hyperpolarization of the cell membrane. This hyperpolarization inactivates the L-type calcium channels, resulting in a decreased Ca²⁺ influx and subsequent smooth muscle relaxation.

   

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Fig. 6.   

Activation pathways in penile smooth muscle. According to Berridge (2008), for example, noradrenaline has two two main actions. It generates IP₃, which activates a cytosolic Ca²⁺ oscillator. It also activates the Rho/Rho kinase signaling pathway to increase the Ca²⁺ sensitivity of the contractile machinery. In addition, the Ca²⁺ transients activate Ca²⁺-sensitive chloride channels that result in membrane depolarization to activate voltage-operated channels. This introduces Ca²⁺ to modulate the oscillator and also creates a flow of current to entrain the oscillatory activity of neighboring cells to account for the way these corpora cavernosa cells contract in near-unison with each other. A, agonist; R, receptor; PLC, phospholipase C; DAG, diacylglycerol; CPI-17, protein-kinase C-potentiated myosin phosphatase inhibitor; SR, sarcoplasmic reticulum; CIC, calcium-induced calcium release; RyR, ryanodine receptor.

According to Berridge (2008), the rhythmical contractions of CC smooth muscle depend on an endogenous pacemaker driven by a cytosolic Ca²⁺ oscillator that is responsible for the periodic release of Ca²⁺ from the sarcoplasmic reticulum (intracellular compartment for Ca²⁺ storage). The periodic pulses of Ca²⁺ often cause membrane depolarization; this is not part of the primary activation mechanism, but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

The major mechanisms involved in smooth muscle contractions, not associated with changes in membrane potential, are the release of IP₃ and the regulation of Ca²⁺ sensitivity. Both mechanisms may be important for the activation of CC smooth muscle. With regard to the physiologically important phosphatidylinositol cascade, many agonists (e.g., α₁-AR agonists, ACh, angiotensins, vasopressin) bind to specific membrane-bound receptors that are coupled to phosphoinositide-specific phospholipase C via GTP-binding proteins. Phospholipase C then hydrolyzes phosphatidylinositol 4,5-biphosphate to 1,2-diacylglycerol (this activates PKC) and IP₃. The water-soluble IP₃ binds to its specific receptor on the membrane of the sarcoplasmic reticulum, thereby opening this Ca²⁺ channel. Because the Ca²⁺concentration in the sarcoplasmic reticulum is approximately 1 mM, Ca²⁺ is thus driven into the sarcoplasm by the concentration gradient, triggering smooth-muscle contraction. This increase in sarcoplasmic Ca²⁺ concentration may activate a distinct Ca²⁺ release channel of the sarcoplasmic reticulum (i.e., the ryanodine receptor-operated channel), leading to a further increase in the Ca²⁺concentration of the sarcoplasm (Somlyo and Somlyo, 1994, 2000; Karaki et al., 1997).

As in striated muscle, the amount of intracellular free Ca²⁺ is the key to regulation of smooth muscle tone. In the resting state, the level of sarcoplasmic free Ca²⁺ amounts to approximately ≈100 nM, whereas in the extracellular fluid, the level of Ca²⁺ is in the range of 1.5 to 2 mM. The cell-membrane Ca²⁺ pump and the Na⁺/Ca²⁺ exchanger maintain this 10,000-fold gradient. Neuronal or hormonal stimulation can open Ca²⁺ channels, resulting in Ca²⁺ entry to the sarcoplasm down its concentration gradient. A rather modest increase in the level of free sarcoplasmic Ca²⁺ by a factor of 3 to 5 up to 550 to 700 nM then triggers myosin phosphorylation and subsequent smooth-muscle contraction.

In the smooth muscle cell, Ca²⁺ binds to calmodulin, which is in contrast to striated muscles, where intracellular Ca²⁺ binds to the thin-filament-associated protein troponin (Chacko and Longhurst, 1994; Karaki et al., 1997). The Ca²⁺-calmodulin complex activates myosin light-chain kinase (MLCK) by association with the catalytic subunit of the enzyme. The active MLCK catalyzes the phosphorylation of the regulatory light-chain subunits of myosin (MLC₂₀). Phosphorylated MLC₂₀ activates myosin ATPase, thus triggering cycling of the myosin heads (cross-bridges) along the actin filaments, resulting in contraction of the smooth muscle. A decrease in the intracellular level of Ca²⁺ induces a dissociation of the Ca²-calmodulin MLCK complex, resulting in dephosphorylation of the MLC₂₀ by myosin light-chain phosphatase and in relaxation of the smooth muscle (Somlyo and Somlyo, 1994, 2000; Karaki et al., 1997; Hirano, 2007).

In CC smooth muscle, which unlike most smooth muscles spends the majority of its time in the contracted state, an overall myosin isoform composition was found that was intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasic-like characteristics, respectively (DiSanto et al., 1998; Berridge, 2008). Smooth-muscle myosin is composed of a pair of myosin heavy chains and two pairs of myosin light chains (MLC17 and MLC20) that are intimately intertwined. It has been shown that myosin heavy chain pre-mRNA is alternatively spliced to generate isoforms known as SM-A and SM-B. The SM-B isoform is predominantly found in SMs that demonstrate a more phasic contractile nature (e.g., urinary bladder), whereas the SM-A isoform is found in more tonic-type SM (e.g., aorta). DiSanto et al. (1998) have demonstrated that the CC smooth muscle possesses a myosin isoform composition somewhat intermediate between bladder and aortic SM. Zhang et al. (2009b) investigated the effects of blebbistatin, a small cell-permeable molecule originally reported as a selective inhibitor of myosin II isoforms. Blebbistatin completely relaxed human CC precontracted with phenylephrine in a dose-dependent manner. Maximal ICP and ICP/mean arterial pressure were dose-dependently increased by intracavernosal blebbistatin injections. These results of Zhang et al., (2009b) suggested an important role for the smooth contractile apparatus in the molecular mechanism of erection and suggested the possibility of blebbistatin binding at myosin II as a therapeutic treatment for ED by targeting SM contractile pathways.

In smooth muscle, the force/Ca²⁺ ratio is variable and depends partly on specific activation mechanisms. For example, α-AR agonists induce a higher force/Ca²⁺ ratio than does a depolarization-induced increase (i.e., KCl) in intracellular Ca²⁺, suggesting a “Ca²⁺-sensitizing” effect of agonists. Furthermore, it has been shown that at a constant sarcoplasmic Ca²⁺ level, decrease of force (“Ca²⁺-desensitization”) can be observed. It has generally been accepted that the key player in “calcium sensitization” is the MLC₂₀ phosphorylation-dependent mechanism. Thus the balance between pathways leading to an increase in MLC₂₀ phosphorylation and those leading to a decrease in MLC₂₀ phosphorylation determine the extent of the “calcium sensitization” (Hirano, 2007).

2. Relaxation.

As in other smooth muscles, CC smooth muscle relaxation is mediated via the intracellular cyclic nucleotide/protein kinase messenger systems. Via specific receptors, agonists activate membrane-bound adenylyl cyclase, which generates cAMP. cAMP then activates protein kinase A (cAK) and, to a lesser extent, cGK. ANF acts via pGC (Lucas et al., 2000), whereas NO stimulates sGC; both generate cGMP, which activates cGKI and, to a lesser extent, cAK. Activated cGKI and cAK phosphorylate phospholamban, a protein that normally inhibits the Ca²⁺ pump within the membrane of the sarcoplasmic reticulum. The Ca²⁺ pump is then activated and, consequently, the level of free cytoplasmic Ca²⁺ is reduced, resulting in smooth-muscle relaxation. Likewise, the protein kinases activate the cell-membrane Ca²⁺ pump, leading to a decreased sarcoplasmic Ca²⁺ concentration and to subsequent relaxation (Somlyo and Somlyo, 1994, 2000; Karaki et al., 1997; Berridge, 2008). Hashitani et al. (2002) suggested that decreasing the sensitivity of contractile proteins to Ca²⁺ may be the key mechanism of NO-induced relaxation in CC smooth muscle. In CC smooth muscle from the guinea pig, they found that in NA precontracted preparations, the NO donor SIN-1 inhibited 80% of the contraction and decreased [Ca²⁺]_i by 20%. In contrast, the calcium antagonist nifedipine, reduced [Ca²⁺]_i by 80%, whereas the level of contraction was decreased by only 20%. In high-potassium precontracted preparations, SIN-1 inhibited 80% of the contraction and reduced [Ca²⁺]_i by 20%.

1. Endothelial Dysfunction.

Endothelial dysfunction may be a main underlying factor for ED associated with many risk factors, such as hypertension, dyslipidemia, diabetes, depression, obesity, cigarette smoking, and the metabolic syndrome. Because a systemic endothelial dysfunction may functionally manifest itself early in the penile endothelium, the possibility arises that ED may be an early indicator of CV disease (Jackson et al., 2010; Shin et al., 2011).

2. Aging.

Aging is an important risk factor for ED, and it has been estimated that 55% of men have ED at the age of 75 (Melman and Gingell, 1999; Johannes et al., 2000). Although age-related ED is attributed largely to increased oxidative stress and endothelial dysfunction in the penis, the molecular mechanisms underlying this effect are not fully defined. Aging in humans is also associated with several changes in arterial structure and function, part of them related to the decline of circulating levels of steroids (i.e., testosterone and estradiol) (Buvat et al., 2010). Such changes may be partly responsible for the lack of efficacy of ED treatments. There is evidence for involvement of the NO/cGMP system. Thus, Garban et al. (1995) found that the soluble NOS activity decreased significantly in penile tissue from senescent rats. Lower NOS mRNA expression was found in old rats than in young rats (Dahiya et al., 1997). In another rat model of aging, the number of NOS-containing nerve fibers in the penis decreased significantly, and the erectile response to both central and peripheral stimulation decreased (Carrier et al., 1997). In the aging rabbit, endothelium-dependent CC relaxation was attenuated; however, eNOS was up-regulated both in vascular endothelium and corporal smooth muscle (Haas et al., 1998). Johnson et al. (2011) evaluated whether eNOS uncoupling in the aged rat penis is a contributing mechanism. Their findings suggested that aging induces eNOS uncoupling in the penis, resulting in increased oxidative stress and ED.

3. Diabetes Mellitus.

Diabetes mellitus is a significant risk factor for the development of ED (Saenz de Tejada and Goldstein, 1988; Melman and Gingell, 1999; Johannes et al., 2000; Saigal et al., 2006; Chitaley et al., 2009). According to the Massachusetts Male Aging Study, men with diabetes have a 28% prevalence of ED compared with 9.6% in the general population (Feldman et al., 1994). Men with diabetes have a 75% lifetime risk of developing ED and have earlier onset of ED compared with nondiabetics (Saigal et al., 2006). Many factors can contribute to diabetes-induced ED. Systemic effects of hyperglycemia and hypogonadism contribute to the development of impaired vasodilatory signaling, smooth muscle cell hypercontractility, and veno-occlusive disorder (Hidalgo-Tamola and Chitaley, 2009; Malavige and Levy, 2009). In isolated CC from patients with diabetes and ED, both neurogenic and endothelium-dependent relaxation was impaired (Saenz de Tejada et al., 1989); this was also found in rabbits in which diabetes was induced by alloxan (Azadzoi and Saenz de Tejada, 1992). Penile NOS activity and content were reduced in rat models of both type I and II diabetes with ED (Vernet et al., 1995). However, in rats with streptozotocin-induced diabetes, NOS binding increased (Sullivan et al., 1996), and NOS activity in penile tissue was significantly higher than in control rats, despite a significant degradation of mating behavior and indications of defective erectile potency (Elabbady et al., 1995). In humans, diabetic ED was suggested to be related to the effects of advanced glycation end products on NO formation (Seftel et al., 1997). The ability of diabetic tissue to convert l-arginine to l-citrulline via NOS was shown to be reduced, and it was suggested that an increased expression of arginase II in diabetic CC tissue may contribute to the ED associated with this disease (Bivalacqua et al., 2001b). Supporting this view, arginase II isoform deletion was shown to improve CC relaxation in mice with type I diabetes (Toque et al., 2011).

4. Atherosclerosis.

Atherosclerosis is a significant risk factors involved in the development of vasculogenic ED. There is evidence of a strong link between ED and atherosclerosis (Maas et al., 2002; Grover et al., 2006; Jackson et al., 2006, 2010). ED and atherosclerosis share similar risk factors, and both conditions are characterized by endothelial dysfunction and impaired NO bioavailability. Recent data suggest that ED may serve as a sentinel marker that precedes the clinical diagnosis of atherosclerotic vascular disease (Montorsi et al., 2003; Gazzaruso et al., 2008). ED is an independent predictor of future adverse CV events; many men experience symptoms of ED years before their first diagnosis of CV disease. In a rabbit model of atherosclerotic ED (Azadzoi and Goldstein, 1992; Azadzoi et al., 1996), it was shown that chronic cavernosal ischemia impaired not only endothelium-dependent but also neurogenic CC relaxation and NOS activity (Azadzoi et al., 1998). There was also an increased output of constrictor eicosanoids in the CC. l-Arginine administration failed to improve CC relaxation, which was suggested to be due to impairment of the NOS activity and reduction of NO formation.

5. Hypercholesterolemia.

Hypercholesterolemia was also found to impair endothelium-mediated relaxation of rabbit CC smooth muscle (Azadzoi and Saenz de Tejada, 1991; Azadzoi et al., 1998). Hypercholesterolemia did not affect NOS activity, but impaired endothelium-dependent but not neurogenic relaxation of rabbit CC tissue. Because the endothelium-dependent relaxation was improved after treatment with l-arginine, it was speculated that there was a deficient NO formation because of lack of availability of l-arginine in the hypercholesterolemic animals (Azadzoi and Saenz de Tejada, 1991; Azadzoi et al., 1998).

6. Smoking.

Smoking is a major risk factor in the development of erectile dysfunction (Mannino et al., 1994; Gades et al., 2005; Shiri et al., 2005; Tostes et al., 2008). Clinical and basic science studies provide strong indirect evidence that smoking may affect penile erection by the impairment of endothelium-dependent smooth muscle relaxation or more specifically by affecting NO production via increased ROS generation. Whether nicotine or other products of cigarette smoke mediate all effects related to vascular damage is still unknown (Tostes et al., 2008).

7. Radical Prostatectomy.

Despite advances in surgical technique, ED after radical prostatectomy, which remains the standard treatment for men with clinically localized prostate cancer, is a common complication. This is mainly attributed to temporary cavernosal nerve damage resulting in penile hypoxia, smooth muscle apoptosis, fibrosis, and veno-occlusive dysfunction (Magheli and Burnett, 2009).

1. Phosphodiesterase Inhibitors.

Current ED treatment guidelines recommend PDE5 inhibitors as the first-line treatment (Hatzimouratidis et al., 2010). All the “major” PDE5 inhibitors, sildenafil, tadalafil, and vardenafil, have been assessed as effective and safe (Hatzimouratidis and Hatzichristou, 2008; Eardley et al., 2010). The overall efficacy rate of these drugs is approximately 60 to 70% but is considerably lower in some patient populations, such as those with severe neurological damage, ED after radical prostatectomy, diabetes, or severe vascular disease (Hatzimouratidis and Hatzichristou, 2008). The choice of a PDE5 inhibitor depends on several factors, including the frequency of intercourse and the patient’s personal experience with the agent (Mirone et al., 2009).

PDE5 inhibitors were initially introduced as on-demand treatment; however, tadalafil has also been approved for continuous, everyday use in 2.5- and 5-mg doses. The actions of PDE5 inhibitors are often described in terms of selectivity (PDE5 versus other PDEs) and potency (the concentration needed for effect). PDE5 inhibitor selectivity is a key factor determining its adverse effect profile and may vary between agents (Table 1). Sildenafil and vardenafil cross-react slightly with PDE6. Because PDE6 predominates in the retina, this may explain the complaint of some patients that sildenafil or vardenafil may cause visual disturbances (< 2% of patients). Tadalafil to some extent cross-reacts with PDE11 (found in, for example, the heart, testes, and the anterior pituitary), but the consequences of this effect are unknown.

The common pharmacokinetic properties of PDE5 inhibitors [e.g., bioavailability, maximum plasma concentration (C_max), time (T_max) required for attaining C_max, and time required for elimination of half the inhibitor from plasma (t_1/2)] all influence efficacy (Table 2) (Gupta et al., 2005). Sildenafil, vardenafil, udenafil, and avanafil have broadly similar T_max, which predicts a similar time of onset of action. The t_1/2 values of tadalafil and udenafil are longer than those of the other PDE5 inhibitors, which could be caused by the slower intestinal absorption and/or slower degradation of these drugs by the liver, or by other factors. The extended t_1/2 of tadalafil provides a longer therapeutic effect, and this may also be the case for udenafil and SLx-2101. The C_max of vardenafil is significantly lower than that for sildenafil and tadalafil, probably depending on the lower bioavailability (Gupta et al., 2005). PDE5 inhibitors are degraded in the liver, and interactions with ketoconazole, for example (inhibiting CYP3A4) may prolong their effect duration. The duration of effect of a PDE5 inhibitor is not always reflected in its elimination from plasma. Molecular mechanisms that can contribute to this have been suggested (Francis et al., 2008). Thus, there may be a persistence of biochemical effects after the inhibitor is cleared from cells (memory effect). In addition, because inhibitors bind tightly to PDE5 in, e.g., muscle cells, this could significantly retard their exit from these cells and prolong their effects (Francis et al., 2008).

Common adverse events with PDE inhibitors include headache (10–16%), flushing (5–12%), dyspepsia (4–12%), nasal congestion (1–10%), and dizziness (2–3%) (Hatzimouratidis et al., 2010). Tadalafil may cause back pain/myalgia in 6% of patients. Adverse events are generally mild in nature and self-limited by continuous use, and the dropout rate due to adverse events is similar to that seen with placebo. Clinical trials and postmarketing data of all PDE5 inhibitors have demonstrated the drugs to be safe in patients with CV disease. Thus, no increase in myocardial infarction rates has been observed (Vlachopoulos et al., 2009). No PDE inhibitor has adversely affected total exercise time or time to ischemia during exercise testing in men with stable angina. In fact, they may improve exercise tests. PDE5 inhibitors may even be beneficial in CV disease (Takimoto et al., 2005), and sildenafil has been approved for treatment of pulmonary arterial hypertension (Galiè et al., 2005).

Nitrates are totally contraindicated with all PDE inhibitors due to unpredictable hypotension. The duration of interaction between organic nitrates and PDE inhibitors varies according to the PDE inhibitor and the nitrate. If a patient develops angina while using a PDE inhibitor, other antiangina agents may be used instead of nitroglycerin or until the appropriate time has passed (24 h for sildenafil or vardenafil and 48 h for tadalafil) (Vlachopoulos et al., 2009).

2. Prostaglandin E₁.

Although PDE5 inhibitor remains the most common initial therapy in men with erectile dysfunction, intraurethral alprostadil may be a reasonable treatment option for sildenafil nonresponders (Jaffe et al., 2004), particularly in men having undergone prior radical retropubic prostatectomy (McCullough, 2001; McCullough et al., 2010). Vasoactive agents can be administered topically to the urethral mucosa and can apparently be absorbed into the corpus spongiosum and transferred to the CC. Retrograde filling of the cavernosal bodies through the deep dorsal vein and its circumflex branches seems to be the most relevant way of drug transfer after intraurethral application of prostaglandin E₁ (Bschleipfer et al., 2004).

PGE₁ (alprostadil) and a PGE₁/prazosin combination was demonstrated to produce erections in a majority of patients with chronic organic ED (Peterson et al., 1998). In a prospective, multicenter, double-blind, placebo-controlled study on 68 patients with long-standing ED of primarily organic origin (Hellstrom et al., 1996), transurethrally administered alprostadil produced full enlargement of the penis in 75.4%, and 63.6% of the patients reported intercourse. The most common side effect was penile pain, experienced by 9.1 to 18.3% of the patients receiving alprostadil. There were no episodes of priapism. In another double-blind, placebo-controlled study on 1511 men with chronic ED from various organic causes, 64.9% had intercourse successfully when taking transurethral alprostadil compared with 18.6% on placebo (Padma-Nathan et al., 1997). Again, the most common side effect was mild penile pain (10.8%).

For men who find intracavernosal injections problematic, the ease of intraurethral administration of alprostadil is an option (Nehra et al., 2002). As mentioned, intraurethral alprostadil may also be an option in men having undergone prior radical retropubic prostatectomy (McCullough, 2001; McCullough et al., 2010). Penile pain remains a problem in many patients.

3. Organic Nitrates.

Nitroglycerin and other organic nitrates are believed to cause smooth muscle relaxation by stimulating soluble GC via enzymatic liberation of NO (Feelisch, 1992); theoretically, this seems to be a logical way to improve erection in patients with ED. Both nitroglycerin and isosorbide nitrate were found to relax isolated strips of human CC (Heaton, 1989). The observation that topical application of nitroglycerin to the penis may lead to erection adequate for sexual intercourse (Talley and Crawley, 1985) stimulated several investigations on the efficacy of this potential mode of treatment of ED. Despite effectiveness in patients with ED, demonstrated in several placebo-controlled studies (Andersson, 2001), the effect of transdermal nitroglycerin is limited, and this treatment does not seem to be a viable alternative today. As underlined previously, organic nitrates are contraindicated in men taking PDE5 inhibitors.

4. K⁺-Channel Openers.

Several K⁺-channel openers (pinacidil, cromakalim, lemakalim, and nicorandil) have been shown to be effective in causing relaxation of isolated cavernosal tissue from both animals and man and to produce erection when injected intracavernosally in monkeys and humans (Andersson, 2001). However, only minoxidil, an arteriolar vasodilator used as an antihypertensive agent in patients with severe hypertension, seems to have been tried as an oral treatment in man. The clinical experiences with the drug are limited (Andersson, 2001), and K⁺-channel opening with the drug, even if it may work in some patients, has not been proven in controlled clinical trials to be a viable option in men with ED. No new developments have been reported during the last decade.

5. α-Adrenoceptor Antagonists.

6. Opioid Receptor Antagonists.

It is well documented that long-term injection of opioids can lead to decreased libido and ED (Parr, 1976; Crowley and Simpson, 1978; Mirin et al., 1980; Abs et al., 2000; Hallinan et al., 2008), possibly because of hypogonadotropic hypogonadism (Mirin et al., 1980; Abs et al., 2000; Hallinan et al., 2008; Vuong et al., 2010). Assuming that endogenous opioids may be involved in sexual dysfunction, opioid receptor antagonists have been suggested to be effective as a treatment (Fabbri et al., 1989; Billington et al., 1990). There are some positive clinical experiences with naltrexone, which together with its active metabolite 6-β-naltrexol are competitive antagonists at μ- (and κ-opioid) receptors. This may illustrate the proof of principle (Andersson, 2001), and it cannot be excluded that increased inhibition by opioid peptides may be a factor contributing to nonorganic erectile failure in some patients. In such patients, naltrexone therapy may be a useful therapeutic agent. However, well controlled studies confirming this are lacking, and there seems to have been no new developments during the last decade.

7. Apomorphine.

Apomorphine, a dopamine receptor agonist that stimulates both dopamine D₁– and D₂– like receptors, has been shown to induce penile erection in rats (Mogilnicka and Klimek, 1977; Benassi-Benelli et al., 1979) as well as in healthy men (Lal et al., 1984) and in men with ED (Lal et al., 1987, 1989). l-DOPA may also stimulate erection in patients with Parkinson’s disease (see, e.g., Vogel and Schiffter, 1983). It has been suggested that dopamine D₂ receptor stimulation may induce penile erection in rats, whereas activation of D₁ receptors have the opposite effect (Zarrindast et al., 1992). In rhesus monkeys, quinelorane, a dopamine D₂ receptor agonist produced penile erection (Pomerantz, 1991), favoring the view that D₂ receptor stimulation is important for this response. Apomorphine has a higher affinity for the D₂– than D₁-like receptors (Rampin et al., 2003). D₂ receptors in the PVN are believed to be the main site for the induction of erections in the rat (Chen et al., 1999). This may also be the case in man (Brien et al., 2002).

Heaton et al. (1995) reported that apomorphine, absorbed through the oral mucosa, will act as an erectogenic agent. This has been largely confirmed in RCTs, and there is evidence that apomorphine is efficacious in the treatment of erectile dysfunction in the broad ED population at doses of 2 and 3 mg taken sublingually in an on demand fashion (Dula et al., 2000, 2001; Heaton et al., 2002; Von Keitz et al., 2002). The tolerability of apomorphine at a dose of 2 and 3 mg has been well investigated (Fagan et al., 2001; Adams et al., 2002; Ralph et al., 2002). The most common side effects are nausea, headache, and dizziness, with small numbers of patients developing syncope. This latter side effect was particularly noted at doses higher than those licensed for use in Europe. Overall, providing that the drug is used in line with labeling, there seems to be no evidence of significant tolerability of safety issues (Eardley et al., 2010).

Although the drug has shown statistically significant benefit over a placebo in the phase II/III clinical trials, examination of the pooled data revealed a low net benefit ratio (i.e., active efficacy minus placebo efficacy) with figures between only 11 and 13% (Stief et al., 2002). This limited efficacy, especially in patients with organic ED, which was confirmed in several studies (Perimenis et al., 2004a,b; Strebel et al., 2004; Gontero et al., 2005), leads to the suggestion that the drug could be best suited for men with mild ED. Subsequent comparative prospective studies between apomorphine SL and sildenafil provided clear evidence that sildenafil is more effective than apomorphine, and the high preference rates were in favor of sildenafil (Porst et al., 2007; Afif-Abdo et al., 2008; Giammusso et al., 2008; Pavone et al., 2008). Because of the superiority of sildenafil, apomorphine SL never reached noteworthy acceptance.

8. Trazodone.

Trazodone is an “atypical” antidepressive agent, chemically and pharmacologically distinct from other currently available antidepressants (Haria et al., 1994). The drug selectively inhibits central 5-HT uptake and increases the turnover of brain dopamine but does not prevent the peripheral reuptake of NA (Georgotas et al., 1982). Trazodone has also been demonstrated to block receptors for 5-HT and dopamine, whereas its major metabolite, m-CCP, has agonist activity at 5-HT_2C receptors (Monsma et al., 1993). This metabolite induces erection in rats and selectively increases the spontaneous firing rate of the cavernosal nerves (Steers and de Groat, 1989). The mode of action of trazodone in depression is not fully understood; it has a marked sedative action. Trazodone has a serum half-life of approximately 6 h and is extensively metabolized (Haria et al., 1994). Trazodone and its major metabolite were shown to have an α-AR blocking effect in isolated human cavernosal tissue (Blanco and Azadzoi, 1987; Saenz de Tejada et al., 1991). Krege et al. (2000) showed trazodone to have high to moderate affinity for human α₁– and α₂-ARs, respectively, and that the drug did not discriminate between subtypes of α₁– and α₂-ARs. The active metabolite, m-CCP, seemed to have no significant peripheral effects.

Orally administered trazodone has been associated with priapism in potent men (Azadzoi et al., 1990) and with increased nocturnal erectile activity in healthy volunteers (Saenz de Tejada et al., 1991). Positive clinical experience with the drug has been reported (Lance et al., 1995). However, in double-blind, placebo-controlled trials in patients with different etiology of their ED, no effect of trazodone (150–200 mg/day) could be demonstrated (Meinhardt et al., 1997; Enzlin et al., 2000).

Even if the information from randomized, controlled clinical trials does not support the view that trazodone is an effective treatment for most men with ED, it cannot be excluded that the drug may be an alternative in some anxious or depressed men. In a pilot study, it was observed that trazodone may be beneficial in the management of selective serotonin-reuptake inhibitor-induced sexual dysfunction (Stryjer et al., 2009). In men with ED and a degree of psychogenic component sufficient to reduce the efficacy of medical management, a combination of trazodone with sildenafil gave promising results in a pilot study (Taneja, 2007).

9. Melanocortin Receptor Agonists.

MT-II is a synthetic cyclic heptapeptide that was initially designed as an artificial tanning agent (King et al., 2007). It is a cyclic nonselective melanocortin receptor agonist that, when injected subcutaneously, was found to be a potent initiator of penile erection in men with nonorganic ED (Wessells et al., 1998, 2000). However, yawning/stretching and in some cases severe nausea and vomiting limited its use.

PT-141 (bremelanotide) is a synthetic heptapeptide; it is a deaminated derivative and likely metabolite of MT-II. This compound has strong binding to MC receptors 1, 3, and 4, with a higher affinity for the MC4 receptor over MC3, where it acts as an agonist (Giuliano, 2004; King et al., 2007).

A number of clinical studies have evaluated the effect of PT-141 (Molinoff et al., 2003; Diamond et al., 2004, 2005; Rosen et al., 2004). PT-141 was administered intranasally at doses ranging from 4 to 20 mg to 32 healthy subjects in a randomized, double-blind, placebo-controlled crossover study (Diamond et al., 2004). This study was also carried out without visual sexual stimulation. Compared with placebo-treated subjects, PT-141 significantly increased erectile activity. The duration of erections with rigidity (Rigi-Scan monitoring) greater than 60% base was approximately 140 min in the subjects treated with 20 mg of PT-141 compared with 21 min in the placebo-treated group.

In a placebo-controlled crossover trial of 24 men with mild to moderate ED (Diamond et al., 2004), the effect of PT-141 (20 mg) was given together with visual sexual stimulation (erotic films). A 3-fold increase in erectile activity was observed in subjects given PT-141 compared with placebo. The duration of erection and penile rigidity were also significantly increased after PT-141 administration.

A randomized, prospective placebo-controlled crossover study compared treatment of 19 patients with ED with sildenafil (25 mg) alone versus sildenafil (25) with 7.5 mg of intranasal PT-141 (Diamond et al., 2005). Coadministration of the two agents resulted in significantly prolonged time of increased base rigidity (>60%) compared with sildenafil alone during a 2.5-h monitoring session. The combination of drugs was well tolerated with no significantly increased side effects over either sildenafil or PT-141 alone. No serious side effects have been reported after PT-141 administration in either normal subjects or in patients with ED.

It is obvious that MC receptor agonists may have effects in patients with ED that can be clinically useful. However, the relation between efficacy and adverse effects has to been determined in large RCTs before regulatory approval and possible clinical introduction.

1. Papaverine.

Intracavernosal papaverine injection was the first clinically effective pharmacological therapy for ED (Virag, 1982). The drug is often classified as a phosphodiesterase inhibitor, but it has a very complex mode of action and may be regarded as a “multilevel acting drug” (Andersson, 1994). It is difficult to establish which of its several possible mechanisms of action predominates at the high concentrations that can be expected when the drug is injected intracavernosally. In vitro, it has been shown that papaverine relaxes the penile arteries, the cavernosal sinusoids, and the penile veins (Kirkeby et al., 1990). In dogs, Juenemann et al. (1986) demonstrated that papaverine had a dual hemodynamic effect, decreasing the resistance to arterial inflow and increasing the resistance to venous outflow. The latter effect, which has been demonstrated also in man (Delcour et al., 1987), may be related to activation by papaverine of a veno-occlusive mechanism. Papaverine is effective but is no longer used as monotherapy because of its high rates of fibrosis and priapism.

2. α-Adrenoceptor Antagonists.

Because NA is considered to be one of the main factors maintaining CC smooth muscle tone by stimulating α-ARs, it could be expected that blocking these receptors would cause an erectile response. However, the experiences α-adrenoceptor antagonists treatment as monotherapy have not been very successful.

3. Prostaglandin E₁ (Alprostadil).

PGE₁, injected intracavernosally, alone or in combination, is today a second-line treatment for ED (Alexandre et al., 2007; Albersen et al., 2011). Several aspects of its effects and clinical use have been reviewed previously (Linet and Ogrinc, 1996; Porst, 1996; Andersson, 2001; Alexandre et al., 2007). In clinical trials, 40 to 70% of patients with ED respond to intracavernosal injection of PGE₁. The reason why many patients do not respond is not known. Angulo et al. (2000) demonstrated that the combination of PGE₁ with S-nitrosoglutathione consistently relaxed penile smooth muscle whether or not it relaxed well to PGE₁. They suggested that the clinical response to PGE₁ may be limited in some patients by the specific lack of response of penile smooth muscle to PGE₁ while maintaining the ability to relax in response to agents that activate alternative relaxant pathways. A combination of PGE₁ and S-nitrosoglutathione had a synergistic interaction to relax penile trabecular smooth muscle, and it was speculated that such a combination may have significant therapeutic advantages in the treatment of male ED. However, there seem to have been no new developments of this combination.

PGE₁ is metabolized in penile tissue to PHE₀ (Hatzinger et al., 1995), which is biologically active and may contribute to the effect of PGE₁. PGE₁ may act partly by inhibiting the release of NA (Molderings et al., 1992), but the main action of PGE₁ and PGE₀ is probably to increase the intracellular concentrations of cAMP in the CC smooth muscle cells through EP receptor stimulation (Palmer et al., 1994; Lin et al., 1995).

PGE₁ is known to have a variety of pharmacological effects. For instance, it produces systemic vasodilation, prevents platelet aggregation, and stimulates intestinal activity. Administered systemically, the drug has been used clinically to a limited extent. Little is known about its pharmacokinetics, but it has a short duration of action and is extensively metabolized. As much as 70% may be metabolized in one pass through the lungs (Golub et al., 1975), which may partly explain why it seldom causes circulatory side effects when injected intracavernosally.

4. Vasoactive Intestinal Polypeptide.

As discussed previously, a role for VIP as neurotransmitter and/or neuromodulator in the penis has been postulated by several investigators, but its physiological importance for penile erection has not been established (Andersson, 2001). However, the inability of VIP to produce erection when injected intracavernosally in potent men (Wagner and Gerstenberg, 1988) or men with ED (Adaikan et al., 1986; Kiely et al., 1989; Roy et al., 1990) indicated that it cannot be the main NANC mediator for relaxation of penile erectile tissues.

VIP has been shown to produce a wide range of effects. It is a potent vasodilator, inhibits contractile activity in many types of smooth muscle, stimulates cardiac contractility, and many exocrine secretions. It stimulates adenylyl cyclase and the formation of cAMP (Palmer et al., 1994; Fahrenkrug, 2001). VIP given intravenously can produce hypotension, tachycardia, and flushing (Frase et al., 1987; Krejs, 1988). However, the plasma half-life of the peptide is short, which may contribute to the fact that systemic side effects are rare when it is administered intracavernosally (McMahon, 1996; Dinsmore et al., 1999; Sandhu et al., 1999).

As mentioned, Wagner and Gerstenberg (1988) showed that VIP, even in a high dose (60 μg), was unable to induce erection on intracavernosal injection in potent men. On the other hand, when used in conjunction with visual or vibratory stimulation, intracavernosal VIP facilitated normal erection. Kiely et al. (1989) injected VIP, papaverine, and combinations of these drugs with phentolamine intracorporally in 12 men with ED of various causes. They confirmed that VIP alone is poor at inducing human penile erections. However, in combination with papaverine, VIP produced penile rigidity similar to that obtained with papaverine and phentolamine. Gerstenberg et al. (1992) administered VIP together with phentolamine intracavernosally to 52 patients with erectile failure. Forty percent of the patients had previously received treatment with papaverine alone or with papaverine together with phentolamine. After sexual stimulation, all patients obtained erection sufficient for penetration. Those patients previously treated with papaverine or papaverine/phentolamine stated that the action of the VIP combination was more like the normal coital cycle. No patient developed priapism, corporal fibrosis, or any other serious complication (Gerstenberg et al., 1992). These positive results have been confirmed by other investigators (McMahon, 1996; Dinsmore and Alderdice, 1998; Dinsmore et al., 1999; Sandhu et al., 1999; Dinsmore and Wyllie, 2008). Thus, Sandhu et al. (1999) found in a prospective, double-blind, placebo-controlled study on 304 patients with psychogenic ED, using a novel auto-injector, that more than 81% of patients and 76% of partners reported an improved quality of life. Similar results were obtained by Dinsmore et al. (1999). On the basis of these clinical trials, both the efficacy and safety of the combination has been confirmed, and the combination has approved for treatment of men with ED in the United Kingdom, Denmark, and New Zealand. Most commonly observed adverse effects were facial flushing and headache.

5. Apomorphine.

In a placebo-controlled, double-blind study on healthy volunteers, it was shown that apomorphine, injected subcutaneously (0.25–0.75 mg), was able to induce erection (Lal et al., 1984). This was confirmed by Danjou et al. (1988), showing that apomorphine induced erection and potentiated the erection induced by visual erotic stimulation. There was no increase in libido, which was in agreement with previous observations (Julien and Over, 1984). In 28 patients with ED, Lal et al. (1989) found that 17 responded with erection after subcutaneous apomorphine (0.25–1.0 g); no erection developed after placebo. Segraves et al. (1991) also administered apomorphine subcutaneously (0.25–1.0 g) to 12 men with psychogenic ED in a double-blind and placebo-controlled study. They found a dose-related increase in maximal penile circumference. An erection with a >1-cm increase in maximal penile circumference was obtained in 11 of the 12 patients.

It cannot be excluded that a subgroup of impotent patients may have an impairment of central dopaminergic functions and that the principle of dopamine receptor stimulation can be used not only diagnostically but also therapeutically. The therapeutic potential of subcutaneous apomorphine, however, seems to be limited mainly because of frequently occurring side effects. High doses (i.e., up to 5–6 mg in adult patients) may cause respiratory depression, and in the low dose range (0.25–0.75 mg) where effects on penile erection can be demonstrated, emesis, yawning, drowsiness, transient nausea, lacrimation, flushing, and dizziness may occur (Lal et al., 1984; Segraves et al., 1991). Lal et al. (1987) observed that nonresponders, but not responders, experienced side effects. However, apomorphine administered subcutaneously does not seem to have an acceptable effect/side effect ratio, and it is no longer used therapeutically.

6. Linsidomine Chlorhydrate and Other NO Donors.

Linsidomine, the active metabolite of the antianginal drug molsidomine, is believed to act by nonenzymatic liberation of NO (Feelisch, 1992). The pharmacology of linsidomine made it an interesting alternative for intracavernosal treatment of ED and preliminary studies seemed promising. However, the initial positive results were not confirmed (Andersson, 2001), and the drug is no longer used therapeutically.

Intracavernosal NO donors such as SNP seem effective to treat ED but have been controversial because of hypotensive side effects (Martinez-Piñeiro et al., 1995; Martínez-Piñeiro et al., 1998; Shamloul et al., 2005). Lasker et al. (2010) showed in the rat that sodium nitrite (NaNO₂), administered intracavernosally, increased ICP, decreased systemic arterial pressure, and was 1000-fold less potent than the NO donor SNP. They suggested that in the rat, NaNO₂ is converted to vasoactive NO in the corpora cavernosum and systemic vascular bed by different mechanisms. Thus, experiments with the NOS inhibitor l-NAME and the xanthine oxidoreductase inhibitor allopurinol suggested that nitrite bioactivation in the corpora was mediated through eNOS, whereas nitrite bioactivation in the systemic vascular beds was due largely to the activity of xanthine oxidoreductase. It was also suggested that the ability of nitrite to enhance erectile activity motivates further investigation in the use of nitrite as a therapeutic agent for ED.

7. Combination Therapy.

Phentolamine, papaverine, PGE₁, and VIP are the vasoactive agents most commonly used in combination therapy to treat ED. In theory, combination therapy may offer better efficacy, because many of the drugs would be assumed to act synergistically but a reduction in incidence of side effects and cost per dose can also be expected. An often-used combination is trimix, a mixture of papaverine, phentolamine, and PGE₁. Bechara et al. (1996) reported better results with the combination than with PGE₁ alone. However, Seyam et al. (2005) compared trimix using a 1-mg dose of phentolamine and different doses of papaverine and PGE₁ with a 20-μg dose of PGE1, found no significant differences in hemodynamic effects, rigidity, pain, and self-satisfaction between the two drugs. However, trimix produced a longer duration of erection and more priapism than PGE₁. This and most other combination therapies remain unlicensed. However, the combination of VIP and phentolamine has been approved in several countries.